The long-term objective of these proposed studies is to establish a completion vitro RNA template to a duplex DNA-nucleoprotein particle that is competent for integration. Our investigation will make use of RNA transcripts, synthesized in vitro, that reproduce the LTRs(long terminal repeats) of the viral genome and essential flanking sequences. Mammalian and synthetic tRNAlys will be isolated to provide natural primers for plus strand synthesis. Methods will be devised to obtain both forms of the HIV reverse transcriptase (p51 and p66) by production in microorganisms that contain the modified cloned reverse transcriptase gene. Other viral and cellular factors will be purified by reconstitution assays. In the requested grant period we will bonus on reconstitution of the tRNA priming, synthesis of the strong stop minus strand intermediate, and minus strand transfer stages of the replicative reaction. The detailed enzymology of these processes will be investigated in order to establish the mechanistic basis for all of these reaction steps. This information should provide additional avenues to the rational design of anti-HIV therapeutic agents.